Nerve agents pose a growing threat to society whether they are released accidentally or deliberately. Current means to counter threats from nerve agents, although temporarily effective, are not adequate. Currently, activated charcoal is used to filter nerve agents from air and water; bleach solution or jet fuel is used for decontaminating protective gear. However, these methods use compositions which have undesirable properties including corrosiveness, flammability, and toxicity. Moreover, these methods can only be used on a small scale, and they are not effective over an extended period of time.
Delivery of active enzyme systems to counter and detoxify chemical and biological warfare agents is a promising and active area of research. While some enzymes in their native form have exhibited effectiveness against nerve agents, there are still many challenges in developing effective detoxification systems, including preservation of high catalytic activity in real conditions, stability of the enzyme system after prolonged storage, suitable means of delivery, and accessability of enzymes to threat agents.
“Detoxifying Nerve Agents”, C&E News Sep. 15, 1997, page 26 reports the current state of the art for detoxification of nerve agents, with special reference to efforts on the part of the U.S. Army. A class of enzymes that is known to catalyze the hydrolysis of organophosphate compounds has been investigated for potential decontamination. The organophosphate anhydrolases (OPAA: EC3.1.8.2) catalyze the hydrolysis of many G-type chemical warfare nerve agents. Specifically, these enzymes have activity against compounds such as sarin, soman, and GF (O-cyclohexyl methylphosphono fluoridate). Covalently linking enzymes to solid substrates and embedding enzymes in polymer matrices are the two most common means for enzyme immobilization. However, the covalent chemistry required for linking an enzyme to a substrate often adversely affects the enzyme's activity. Enzymes embedded in polymer matrices are not accessible freely to the agents present in the surrounding medium.
Branner-Jorgensen, in U.S. Pat. No. 4,266,029, disclose immobilizing enzymes on a mineral oxide which has been coated with gelatin and glutaraldehyde. However, these enzymes are used in fluidized bed operations, and there is no indication that these enzymes can be used to detoxify nerve agents.
Doctor et al., in U.S. Pat. No. 5,366,881, disclose mutant cholinesterase which can be used for detoxifying organophosphates. However, to maintain the activity of the cholinesterases, oximes are added.
Tschang et al., in U.S. Pat. No. 4,461,832, disclose enzymes embedded in silica gel in order to suspend the enzyme. There is no indication that these enzymes retain their activity, or that these enzymes can be used to detoxify nerve agents.
Recently, LeJeune and coworkers reported immobilizing phosphotriesterases onto polyurethane polymers for decontamination purposes (LeJeune et al., Biotechnology and Bioengineering 54:105-114, 1997. However, there are several drawbacks to using polyurethane for immobilizing phosphotriesterases. In addition to being an environmentally unfriendly polymer, polyurethane may not afford the maximal protein stability that can be achieved in the protein's native environment. In addition, the enzymes used in these studies have not been selected for use under field conditions, and suffer many drawbacks, including inhibition by substrate, low turnover, and low stability. Watkins et al., Biological Chemistry 272:25596-25601, 1997) have demonstrated enhanced rate of hydrolysis of phosphorus-fluorine bonds by phosphotriesterases using engineered enzymes.